Allen Reference Atlases One of the primary goals of the Allen Brain Atlas (ABA) is to create a cellular-resolution, genome-wide map of gene expression in the mouse brain. To complement ABA gene expression data, Allen Reference Atlases (ARAs) have been designed and created by Dr. Hong Wei Dong in the coronal and the sagittal plane. The reference atlases are full-color, high-resolution, web-based digital brain atlases accompanied by a systematic, hierarchically organized taxonomy of mouse brain structures. The ABA and ARA data are obtained, using identical methodology, from 8-week old C57Bl/6J male mouse brain(s) prepared as unfixed, fresh-frozen tissue. The ARAs were designed to: (I) Allow users to directly compare gene expression patterns to neuroanatomical structures in the ABA Application (II) Serve as templates for the development of 3D computer graphic models of mouse brain, providing a foundation for the development of informatics based annotation tools (III) Provide a standard neuroanatomical ontology for determining structural annotation and aid in the construction of a detailed searchable gene expression database The coronal ARA consists of 132 coronal sections evenly spaced at 100 µm intervals and annotated to a detail of numerous brain structures. Examples of these images are shown in Figure 1. The sagittal ARA consists of 21 representative sagittal sections spaced at 200 µm, annotated for 71 major brain regions identified at the top level(s) of the brain structure hierarchal tree (Appendix 1). In the sagittal ARA, a number of cell groups are used as landmarks to indicate specific brain levels, i.e. the red nucleus and cranial motor nuclei. Ontology and Brain Structure Tree Development ARA ontology is arranged as a hierarchal organization of brain structures, with prioritization levels identified in the brain structure hierarchal tree (Appendix 1). ARA nomenclature was adopted from the Swanson (2004) rat brain atlas; however the hierarchical organization of the brain structures provided by Swanson has been modified. Nomenclature was also adopted from Hof et al. (2000) mouse brain atlases. The ARAs also referenced two informatics based neuroanatomy resources, the Brain Architecture Management System (BAMS, http://brancusi.usc.edu/bkms/) and BrainInfo (http://braininfo.rprc.washington.edu/menumain.html). In the ARAs three basic divisions of the mouse brain are recognized: (i) cerebral hemispheres, i.e. the cerebrum, endbrain, or telencephalon, consisting of the cerebral cortex and cerebral nuclei; (ii) cerebellum, i.e. the parencephalon, as consisting of cerebellar cortex and cerebellar nuclei; and (iii) brainstem, i.e. the cerebrospinal trunk. The regions are subdivided further, using the nomenclature listed in Appendix 2. The definition of cerebral cortex and cerebral nuclei follows Swanson (2004). The organizational scheme of the brainstem, however, is different from that of Swanson (2004); for the ARA the brainstem is delineated following the classical development scheme of the mammalian brain, consisting of the interbrain (diencephalon), midbrain (mesencephalon), and hindbrain (rhombencephalon). The ARA hindbrain is further subdivided into pons (metencephalon) and medulla (myelencephalon). Within each division of the midbrain, pons, and medulla, brain structures are put into three major categories, sensory related, motor related, and behavioral state related, which are partially compatible with the organization of Swanson (2004). © 2004-2006 Allen Institute for Brain Science. All rights reserved. Page 1 of 20 A B Figure 1 - Sample images from the coronal (A) and sagittal (B) ARA All brain structures annotated in the ARAs are assigned unique colors to visually emphasize their hierarchical positions in the brain. This facilitates the unique definition and segmentation of brain regions. Users can find the hierarchically organized brain structure list (with abbreviations) in the ARA Nomenclature table (Appendix 2). Histology and Photomicrographs A comparison of general methodologies used for the ARAs and two other published mouse brain atlases (Paxinos and Franklin, 2003; Hof et al., 2000) are shown in Appendix 3. Unfixed, fresh-frozen C57Bl/6J © 2004-2006 Allen Institute for Brain Science. All rights reserved. Page 2 of 20 mouse brains were sectioned at 25 µm thickness for the ARAs using the same methods described for the ABA in situ hybridization process. The Nissl staining protocol used was modified from that of Paxinos and Watson (1998) and is described in Appendix 4. Nissl-stained sections were scanned at high resolution (10X, 0.95 µm/pixel) using a Leica DC 500 CCD camera mounted onto a Leica DM 6000 microscope. Annotation of 2D Nissl Sections Digital Nissl stained images were imported into Adobe Photoshop and contrast adjustments were made to the drawing. Annotated maps based on these images were then drawn using Adobe Illustrator CS. Displaying the Allen Reference Atlases The ARA is displayed through the ABA Application (http://www.brain-map.org/). In order to facilitate the use of the ARAs images of the annotated reference sections are presented side-by-side with images of in situ hybridization (ISH) gene expression data. The image viewer is designed to present the reference atlas image that most closely matches the plane of section of the ISH data being viewed. The coronal ARA is displayed with half of the brain presented as a Nissl image, mirrored by the corresponding annotated drawing. The sagittal ARA is displayed to allow users to view the annotated map and Nissl images separately. Users have the option to toggle between the Nissl images and corresponding annotation. The Application provides a virtual microscope for the in-situ hybridization data and the ARAs by allowing magnification, “zoomification”, of the atlas images. References Hof, P.R. and Young, W.G. (2000) Comparative Cytoarchitectonic Atlas of the C57BL/6 and 129 Sv Mouse Brains. Elsevier, Amsterdam. Paxinos, G. and Franklin, K.B.J. (2004) The Mouse Brain in Stereotaxic Coordinates: Compact Second Edition. Elsevier Academic Press, San Diego, CA. Paxinos, G. and Watson, C. (1998) The Rat Brain in Stereotaxic Coordinates (Fourth Edition). Academic Press, San Diego, CA. Swanson, L. (2004) Brain Maps: Structure of the Rat Brain (Third Edition). Elsevier Academic Press, San Diego, CA. © 2004-2006 Allen Institute for Brain Science. All rights reserved. Page 3 of 20 Appendix 1 - ARA Brain Structure Tree © 2004-2006 Allen Institute for Brain Science. All rights reserved. Page 4 of 20 Appendix 2 - ARA Nomenclature 1. Cereberum [cerebral hemisphere, endbrain, telecephalon] (CH) 1.1. Cerebral cortex (CTX) 1.1.1. Cortical plate (CTXpl) 1.1.1.1. Isocortex (ISO)* Frontal pole, cerebral cortex (FRP) Somatomotor areas (MO) Primary motor area (MOp) Secondary motor area (MOs) Somatosensory areas (SS) Primary somatosensory area (SSp) Primary somatosensory area, nose (SS-n) Primary somatosensory area, barrel field (SSp-bfd) Primary somatosensory area, lower limb (SSp-ll) Primary somatosensory area, mouth (SSp-m) Primary somatosensory area, upper limb (SSp-ul) Primary somatosensory area, trunk (SS-tr) Supplemental somatosensory area (SSs) Infralimbic area (ILA) Gustatory areas (GU) Visceral area (VISC) Auditory areas (AUD) Dorsal auditory area (AUDd) Primary auditory area (AUDp) Posterior auditory area (AUDpo) Ventral auditory area (AUDv) Visual areas (VIS) Anterolateral visual area (VISal) Anteromedial visual area (VISam) Lateral visual area (VISl) Primary visual area (VISp) Posterolateral visual area (VISpl) posteromedial visual area (VISpm) Rostrolateral visual area (VISrl) Anterior cingulate area (ACA) Anterior cingulate area, dorsal part (ACAd) Anterior cingulate area, ventral part (ACAv) Prelimbic area (PL) Orbital area (ORB) Orbital area, lateral part (ORBl) Orbital area, medial part (ORBm) Orbital area, ventral part (ORBv) Orbital area, ventrolateral part (ORBvl) Agranular insular area (AI) Agranular insular area, dorsal part (AId) Agranular insular area, posterior part (AIp) Agranular insular area, ventral part (AIv) Retrosplenial area (RSP) Retrosplenial area, lateral agranular part (RSPagl) Retrosplenial area, dorsal part (RSPd) Retrosplenial area, ventral part (RSPv) Posterior parietal association areas (PTLp) Temporal association areas (TEa) Perirhinal area (PERI) Ectorhinal area (ECT) * The six-layered scheme of the isocortex has been recognized almost 100 years (Brodmann, 1909; Zilles and Wree, 1995). These layers are named from superficial to deep: 1, molecular layer (ISO1); 2, superficial supragranular pyramidal layer (ISO2); 3. deep supragranular pyramidal layer (ISO3); 4, granular layer; 5, infragranular pyramidal layer; 6, polymorph layer. Per Swanson (2004), layer 6 is subdivided into two layers, 6a & 6b. The 6b is assigned separately from other cortical layers into the cortical subplate, while other cortical layers remain in the cortical plate (Alvarez-Bolado and Swanson, 1996). © 2004-2006 Allen Institute for Brain Science. All rights reserved. Page 5 of 20 Appendix 2 - ARA Nomenclature 1.1.1.2. Olfactory areas (OLF) Main olfactory bulb (MOB) Main olfactory bulb, glomerular layer (MOBgl) Main olfactory bulb, granule layer (MOBgr) Main olfactory bulb, inner plexiform layer (MOBipl) Main olfactory bulb, mitral layer (MOBmi) Main olfactory bulb, outer plexiform layer (MOBopl) Accessory olfactory bulb (AOB) Accessory olfactory bulb, glomerular layer (AOBgl) Accessory olfactory bulb, granular layer (AOBgr) Accessory olfactory bulb, mitral